Ocular Immunology & Inflammation, 2015; 23(1): 47–52 ! Informa Healthcare USA, Inc. ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.981550
ORIGINAL ARTICLE
devR PCR for the Diagnosis of Intraocular Tuberculosis Pankaj Kataria, MS1, Abiraj Kumar, MS1, Reema Bansal, MS1, Aman Sharma, Vishali Gupta, MS1, Amod Gupta, MS1, Ramandeep Singh, MS1, and Kusum Sharma, MD3
2
MD
,
1
2
Advanced Eye Center, Post Graduate Institute of Medical Education and Research, Chandigarh, India, Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India, and 3Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
ABSTRACT Purpose: To compare the efficacy of devR and MPB64 PCR in the diagnosis of intraocular tuberculosis. Methods: Prospective, nonrandomized study. Seventy-five patients were enrolled in 3 groups. Group A had 25 patients with presumed intraocular tubercular uveitis, group B had 25 controls with specific uveitis other than tubercular uveitis, and group C included 25 non-uveitic negative controls. The undiluted vitreous/ aqueous samples were collected and subjected to PCR assay for devR and MPB64 gene sequence of Mycobacterium tuberculosis (MTB) to detect sensitivity and specificity. Results: devR PCR was positive in 16 (64%) out of 25 patients with presumed tubercular uveitis. MPB64 PCR was positive in 18 (72%) out of 25 patients with presumed tubercular uveitis. The sensitivity and specificity of devR were 64 and 100%, respectively. The sensitivity and specificity of MPB64 PCR were 72 and 100%, respectively. Conclusion: devR PCR is not a better tool than MPB64 PCR for diagnosing intraocular tuberculosis. Keywords: devR PCR, MPB64 PCR, polymerase chain reaction, retinal vasculitis, tubercular vasculitis
Diagnosis of intraocular tuberculosis (IOTB) in a tuberculosis (TB) endemic area poses a challenge due to diverse manifestations, its similarity to other uveitis entities, its paucibacillary nature, the difficulty in obtaining samples, lack of adequate samples, and the limitations of current diagnostic tests. Even though highly effective ATT treatment is available, this treatment has few adverse effects warranting some degree of certainty before starting it. It is difficult to establish the diagnosis of IOTB by histologic and microbiologic methods,1,2 and tuberculin skin sensitivity test (TST).3,4 Interferon-gamma release assays are still in primitive stage to diagnose IOTB.5 The polymerase chain reaction (PCR) technique has emerged as a powerful tool for rapid and accurate detection of the mycobacterial genome, with high specificity and a variable sensitivity.6–11
PCR is especially helpful for ocular fluids, as it can be performed with very small sample size. Most of the earlier studies have used IS6110 as a target specific for mycobacterium tuberculosis (MTb) for IOTB.9–15 However, absence or presence of very few copies of IS6110 has been reported in Indian population,12,13 which can result in missed cases suspected of TB. MPB64 gene-based PCR specific for MTB has been well evaluated for diagnosis of IOTB and has been found to be more sensitive than IS6110 PCR.14,15 The devR sequences are universally detected in MTB strains16,17,18 and it has been well evaluated in various extrapulmonary devR PCR has been shown to be highly sensitive and specific in the diagnosis of pulmonary and extra pulmonarytuberculosis.16–21 To the best of our knowledge, no study has earlier evaluated devR gene-based PCR for diagnosis of
Received 16 August 2014; revised 20 October 2014; accepted 22 October 2014; published online 26 November 2014 Correspondence: Dr. Ramandeep Singh, Associate Professor, Advanced Eye Center, Post Graduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh, India 160012. E-mail: [email protected]
47
48 P. Kataria et al. IOTB. Therefore, the present study was undertaken to evaluate the role of devR PCR in the diagnosis of IOTB and compared its efficacy with MPB64 PCR.
MATERIAL AND METHODS The study was conducted at our tertiary eye care center between July 2012 and November 2013. This study adhered to the guidelines in the Declaration of Helsinki. After institutional ethics committee approval and informed consent of the patient, we prospectively enrolled 75 patients into 3 groups in this study. Group A included 25 patients with presumed tubercular uveitis based on clinical signs and corroborative evidence.22 The diagnosis of TB was presumed when the patient had (a), signs of active uveitis such as cells/flare in the anterior chamber, with or without granulomatous keratic precipitates or iris nodules, iris granuloma, vitreous cells, snowballs, snow-banking, retinal vasculitis with/without perivascular choroiditis scars, with/without vitreous hemorrhage or tractional retinal detachment, serpiginous-like choroiditis, choroidal granuloma, or neuroretinitis; (b) corroborative evidence included positive tuberculin skin test (TST) according to the CDC recommendations (TST, 10-mm induration or more at 48–72 h) or positive Quantiferon-TB (QFT-TB) Gold test, or a positive chest x-ray; (c) all known causes of infectious uveitis or known noninfectious uveitis syndromes ruled out. Patients who were receiving anti-tubercular treatment (ATT) at the time of collection of vitreous fluid or received ATT in the past were excluded. Group B included 25 patients as positive controls, i.e. uveitis patients with other nontubercular etiologies, such as viral uveitis and endophthalmitis. Group C included 25 patients with noninflammatory ocular conditions, such as retinal detachment and macular hole, who were undergoing routing pars plana vitrectomy. No additional tests were done for this group of patients. All the enrolled patients underwent detailed ophthalmological examination, including best corrected visual acuity using the Snellen’s chart, intraocular pressure on Goldmann applanation tonometery, and slit-lamp biomicroscopy. Ancillary tests such as fundus photography, fundus fluorescein angiography, spectral domain optical coherence tomography, and ocular ultrasonography were performed as and when required. Group A patients underwent baseline uveitis investigations that included TST, chest x-ray, erythrocyte sedimentation rate, and Treponema palladium hemagglutination (TPHA) tests. In group B patients, investigations were ordered to diagnose the specific etiology, i.e. PCR for viral etiologies and smear and culture examination of the patients with endophthalmitis.
Sample Collection The aqueous humor was aseptically collected with a disposable 25- or 27-gauge needle on a tuberculin syringe under an operating microscope. The vitreous humor was aseptically collected at the beginning of 3-port pars plana vitrectomy and transferred to the laboratory in the cold chamber immediately. Our aim was to procure vitreous or aqueous cells in an ocular sample from the eye in cases other than vitreous hemorrhage.
Technique and Analysis of PCR The DNA was extracted from the fluid sample using a DNAse tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA specimens were stored at 20 C until used. The devR PCR was performed using the forward primer devR1 (50 -ATCTGTTGTCCCGCATGCC-30 ) and reverse primer devR2 (50 -GTCCAGCGCCCACATCT TT-30 ), which amplified a 162–base pair fragment of the devR single copy gene.21 The MPB64 PCR were performed using the forward primer MPB641 (50 -TCCGCTGCCAGTCGTCTTCC-30 ) and reverse primer MPB642 (50 - GTCCTCGCGAGTCTAGGCCA-30 ), which amplified a 240–base pair fragment of the MPB64 single copy gene.23 During DNA extraction and amplification, strict precautions were taken, including separate areas for DNA extraction, reagent preparation, amplification, and product detection, and regular meticulous cleaning of surfaces with 10% hypochlorite. In addition, all the reagents were aliquoted upon arrival in the laboratory. Positive and negative controls were included with each set of reactions. Positive control DNA extracted from H37RV was used and for negative control PCR grade water was used. Statistical Methods The sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the standard formula as follows (positive test: a = disease, b = no disease; negative test: c = disease, d = no disease): Sensitivity = a/(a + c) Specificity = d/(b + d) Positive predictive value = a/(a + b) Negative predictive value = d/(c + d)
RESULTS Group A comprised 25 patients with 20 males and 5 females. Group B had 15 males and 10 females, while group C had 25 negative controls with 18 males and 7 females. The mean age was 32.12 ± 10.27 Ocular Immunology & Inflammation
PCR for Intraocular Tuberculosis (range: 18–52) years in group A, 38.16 ± 19.64 (range: 8–70) years in group B, and 48.4 ± 16.81 (range: 5–68) years in group C. In group A, vitreous fluid samples were collected from 24 patients, i.e. vasculitis vitreous hemorrhage (16), choroiditis (7), intermediate uveitis (1), and panuveitis (1). Undiluted vitreous sample was taken at the start of the vitreous surgery as they all pars palna vitrectomy (PPV) for therapeutic or diagnositic vitrectomy. Therapeutic PPV was done in eyes with vitreoeus hemorrhage and diagnostic PPV was done in rest of the eyes. Aqueous fluid sample was collected from 1 patient with choroiditis. Vitreous fluid samples were collected from all the patients in groups B and C. In group A, all 25 patients had a positive tuberculin skin sensitivity test (induration of410 mm at 72 h) and 3 patients showed evidence of calcified lymph nodes on chest x-ray (Table 1). None of the patients had miliary TB. In group A, patients with presumed tubercular uveitis, devR PCR was positive in 16 patients and negative in 9 patients (Table 1, Figure 1). MPB64 PCR was positive in 18 patients and negative in 7 patients (Table 1, Figure 2). With devR PCR, we found sensitivity and specificity of 64 and 100%, respectively. With MPB64 PCR, we found sensitivity and specificity of 72 and 100%, respectively. Both PCRs were simultaneously positive in 14 cases out of 25. In 6 of the 25 patients, both PCR results were opposite. Five cases were negative for both devR and MPB64
49
PCR (Tables 2, 3). Combined (at least one positive) sensitivity was 80% and specificity was 100%. In control groups B and C, ocular fluid samples were negative in all patients for both devR PCR and MPB64 PCR (Table 3). There were no false-positive cases. Statistically significant difference was found in terms of difference in PCR results in cases and control groups (p5.001). In subgroup analysis of group A, vasculitis with vitreous hemorrhage subgroup, of the 16 patients, 12 were positive for MPB64 PCR and 9 were positive
FIGURE 1. DevR PCR Gel picture: L1- MM, L2- Positive control with bending pattern on gel, l3-Negative control: L4-L6 Positive clinical samples, L7-8; negative samples.
TABLE 1. devR PCR and MPB64 PCR results in group A. S no
Mantoux test
Chest x-ray
devR PCR
MPB64 PCR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Normal Abnormal Abnormal Abnormal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Negative Negative Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Negative Positive Positive Positive Negative Positive Positive Negative Positive
Negative Negative Positive Positive Negative Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive
!
2015 Informa Healthcare USA, Inc.
Diagnosis Vasculitis with vitreous hemorrhage Panuveitis Choroiditis Choroiditis Intermediate uveitis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Choroiditis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Choroidal granuloma with choroiditis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage
50 P. Kataria et al.
FIGURE 2. MPB64 PCR Gel Picture: L1-MM, L2-Positive control, L3-L4, L6: Positive patient DNA, L5-Negative control, L7, 8-Negative patient DNA.
TABLE 2. Detailed results of positive PCR in group A patients. Specific genome results (Group A = 25 samples) devR positive alone MPB64 positive alone Both devR and MPB 64 positive Either devR or MPB64 positive
Number of samples (%) 16 18 14 20
(64%) (72%) (56%) (80%)
for devR PCR. MPB64 PCR was more sensitive than devR PCR (75 vs. 56.25%, respectively) in this subgroup. In the choroiditis subgroup, of 7 the patients with active choroiditis, all were positive for devR PCR (sensitivity 100%) and 6 were positive for MPB64 PCR (sensitivity 85.7%). One patient of choroiditis had co-existent choroidal granuloma that was both devR and MPB64 PCR positive (Table 4).
DISCUSSION Nucleic acid amplification techniques (NAAT), such as PCR, are a robust method for diagnosis of paucibacilliary extrapulmonary tuberculosis. devR PCR has been used to diagnose extraocular tuberculosis, with sensitivity ranging from 46.7 to 98%16–21 and specificity ranging from 66.7 to 100%.16–21 There is no available study in literature regarding the use of devR PCR for diagnosis of IOTB. Halder et al.20 demonstrated high sensitivity (87.6%) and specificity (92%) of devR PCR in the diagnosis of tubercular meningitis (TBM). Chakravorty et al.,16 in their study with devR PCR, demonstrated that sensitivity ranged between 46.7 and 75.5% and the specificity ranged between 66.7 and 100% in three groups of specimens (pleural fluid, pleural tissue, and lymph node). Hallur et al.19 compared IS6110 with devR PCR in cases of
abdominal tuberculosis and showed a higher sensitivity of 80% with devR PCR as compared to 77% with IS6110 PCR. In the present study, devR PCR was found to be highly specific in the diagnosis of IOTB, corroborating the results of previous studies on extraocular tuberculosis, but it lacked sensitivity in detecting intraocular tuberculosis, similar to previously reported studies from other extrapulmonary sites.16–20 In the present study, MPB64 PCR (72%) was found to be more sensitive than devR PCR (64%) in diagnosing IOTB. However, when any of the two gene targets (MPB64, devR) were used, sensitivity for diagnosis of IOTB increased to 80%. Earlier studies using MPB 64 gene target for diagnosis of IOTB have shown sensitivity ranging from 47.8 to 66.66%.14,15,24 A majority of the PCR assays evaluated for diagnosis of TB have targeted the IS6110 gene, as there are multiple copies of IS6110 in the MTb genome (1–25 copies/genome), resulting in higher sensitivity. However, it has been shown that some Indian strains of MTb do not contain IS6110 or have low copy numbers of this gene, thus missing these cases.13 So, there is need to evaluate other targets for rapid diagnosis of ocular tuberculosis as recently published by Balne et al.25 and Sharma et al.15 IS6110 has been used for the diagnosis of IOTB extensively. When compared to previous studies,10,11,23 sensitivity of devR PCR (64%) was greater in our study. The sensitivity of IS6110 PCR is reported between 37 and 58.82%.10,11,23 MPB64 has been reported as 10,000 times more sensitive than IS6110 for the diagnosis of tubercualr uveitis.14 Our study supports this finding. Although clinically strong suspicious cases of tubercular uveitis were enrolled in group A, some turned out to be negative with PCR. So testing for one particular gene may give false-negative PCR result. To overcome this, a novel multitargeted PCR technique18 has been introduced. Sharma et al.18 reported high sensitivity (77.77%) with multitargeted PCR (IS6110, MPB64, protein b) as compared to individual PCR. This approach would be helpful in the diagnosis of the presumed tubercular uveitis with increased sensitivity. In the present study, a combination of two PCR revealed sensitivity of 80%, when either of the two PCR (devR and MPB64) was positive. Hence, these two have the potential to be used for multitargeted PCR. In subgroup analysis, in patients with the vitreous hemorrhage subgroup, we found the sensitivity of MPB64 (75%) to be greater than that of devR PCR (56.25%). For the diagnosis of Eales’ disease using various PCR primers, previous studies had reported sensitivity of 41.6% (Biswas et al.,11 IS6110), 47.8% (Madhvan et al.,14 MPB64), and 57% (Singh et al.,22 MPB64). In choroiditis patients, sensitivity of devR PCR was 100%, while it was 85.7% for MPB 64. Ocular Immunology & Inflammation
PCR for Intraocular Tuberculosis
51
TABLE 3. Sensitivity and specificity of devR and MPB64 PCR in group A patients. Test devR MPB64 Combined (at least one positive)
Test results
Group A (n = 25)
Groups B and C (n = 50)
Positive Negative Positive Negative Positive Negative
14 11 16 9 20 5
– 50 – 50 – 50
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
64
100
100
84.75
72
100
100
87.71
80
100
100
90.90
PPV, positive predictive value; NPV, negative predictive value.
TABLE 4. Subgroup analysis of group-A patients. Etiologies (n = no. of patients)
devR positive (%)
MPB64 positive (%)
9 (56.25) 7 (100) – –
12 (75) 7 (85.71) – –
Vitreous hemorrhage (16) Choroiditis (7) Intermediate uveitis Panuveitis
Gupta et al.23 reported IS6110 PCR positivity in 4 of the 5 eyes of choroidal tuberculosis simulating serpiginous choroiditis. The study was marred by the small number of patients; larger studies could further show the promising role of devR PCR in eyes with choroiditis. Our study has a few limitations, the major one being the small number of patients. In groups B and C, patients were presumed to be nontubercular in origin and investigations related to uveitis workup were not considered necessary in them. The follow-up of patients and assessment of clinical response to treatment was not carried out in this study. There are some known inherent limitations of PCR, i.e. PCR negativity in a few clinically suspected intraocular TB cases, due to the presence of the low number of bacteria or poor lysis of bacteria in the ocular fluids. Sometimes the tough cell wall of M. tuberculosis makes the isolation of target DNA difficult or it could be affected by the presence of inhibitors in the sample. TST and QFT-TB were not carried out in group B and C patients. This could be one of the limitations and future studies can be planned taking these factors in consideration. Use of clinical diagnosis of presumed intraocular TB as the gold standard has its own issues. Culture/microscopy was not used due to inherent problems of low sensitivity and prolonged turn around time. The alternative of favorable response to anti-tubercular treatment in presumed tubercular uveitis was not used in this study. Despite the above limitations, our results clearly show that MPB64 PCR is is amore sensitive diagnostic test for IOTB in comparison to devR PCR. Further, this study for the first time shows the role of devR PCR in the diagnosis of IOTB. The role of devR needs to be !
2015 Informa Healthcare USA, Inc.
evaluated in a large sample size, especially in IOTB patients with ocular finding of choroiditis.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Helm CJ, Holland GN. Ocular tuberculosis. Surv Ophthalmol. 1993;38:229–256. 2. Bodaghi B, LeHoang P. Ocular tuberculosis. Curr Opin Ophthalmol. 2000;11:443–448. 3. Abrams J, Schlaegel Jr TF. The tuberculin skin test in the diagnosis of tuberculous uveitis. Am J Ophthalmol. 1983;96: 295–298. 4. Huebner RE, Schein MF, Bass Jr JB. The tuberculin skin test. Clin Infect Dis. 1993;17:968–975. 5. Kurup SK, Buggage RR, Clarke GL, et al. Gamma interferon assay as an alternative to PPD skin testing in selected patients with granulomatous intraocular inflammatory disease. Can J Ophthalmol. 2006;41:737–740. 6. Shankar P, Manjunath N, Lakshmi R, et al. Identification of mycobacterium tuberculosis by polymerase chain reaction. Lancet. 1990;335:423. 7. Chan CC, Shen D, Tuo J. Polymerase chain reaction in the diagnosis of uveitis. Int Ophthalmol Clin. 2005;45:41–55. 8. Gupta V, Gupta A, Arora S, et al. Simultaneous choroidal tuberculoma and epididymo-orchitis caused by Mycobacterium tuberculosis. Am J Ophthalmol. 2005;140: 310–312. 9. Biswas J, Therese KL, Madhavan HN. Use of polymerase chain reaction in detection of Mycobacterium tuberculosis complex DNA from vitreous sample of Eales’ disease. Br J Ophthalmol. 1999;83:994. 10. Arora SK, Gupta V, Gupta A, et al. Kapoor GS, Sehgal S. Diagnostic efficacy of polymerase chain reaction in granulomatous uveitis. Tubercle Lung Dis. 1999;79:229–233. 11. Gupta V, Arora S, Gupta A, et al. Management of presumed intraocular tuberculosis: possible role of the polymerase chain reaction. Acta Ophthalmol Scand. 1998;76: 679–682. 12. Das S, Paramasivan CN, Lowrie DB, et al. IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with
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pulmonary tuberculosis in Madras, south India. Tubercle Lung Dis. 1995;76:550–554. Chauhan DS, Sharma VD, Parashar D, et al. Molecular typing of Mycobacterium tuberculosis isolates from different parts of India based on IS6110 element polymorphism using RFLP analysis. Indian J Med Res. 2007;125:577–581. Madhavan HN, Therese KL, Gunisha P, et al. Polymerase chain reaction for detection of Mycobacterium tuberculosis in epiretinal membrane in Eales’ disease. Invest Ophthalmol Vis Sci. 2000;41:822–825. Sharma K, Gupta V, Bansal R, et al. Novel multi-targeted polymerase chain reaction for diagnosis of presumed tubercular uveitis. J Ophthalmic Inflamm Infect. 2013;3:25–31. Chakravorty S, Sen MK, Tyagi JS. Diagnosis of extrapulmonary tuberculosis by smear, culture, and PCR using universal sample processing technology. J Clin Microbiol. 2005;43:4357–4362. Chakravorty S, Pathak D, Dudeja M, et al. PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis. FEMS Microbiol Lett. 2006;257:306–311. Haldar S, Chakravorty S, Bhalla M, et al. Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J Med Microbiol. 2007;56:1356–1362.
19. Hallur V, Sharma M, Sethi S, et al. Development and evaluation of multiplex PCR in rapid diagnosis of abdominal tuberculosis. Diag Microbiol Infect Dis. 2013;76:51–55. 20. Singh KK, Muralidhar M, Kumar A, et al. Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J Clin Pathol. 2000;53: 355–361. 21. Haldar S, Sharma N, Gupta VK, Tyagi JS. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol. 2009;58:616–624. 22. Gupta V, Gupta A, Rao NA. Intraocular tuberculosis: an update. Surv Ophthalmol. 2007;52:561–587. 23. Singh R, Toor P, Parchand S, et al. Quantitative polymerase chain reaction for Mycobacterium tuberculosis in socalled Eales’ Disease. Ocul Immunol Inflamm. 2012;20: 153–157. 24. Gupta V, Gupta A, Arora S, et al. Presumed tubercular serpiginous like choroiditis. Ophthalmology. 2003;110: 1744–1749. 25. Balne PK, Modi RR, Choudhary N, et al. Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis. J Ophth Inflam Infec. 2014;4:10–15.
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ORIGINAL ARTICLE
devR PCR for the Diagnosis of Intraocular Tuberculosis Pankaj Kataria, MS1, Abiraj Kumar, MS1, Reema Bansal, MS1, Aman Sharma, Vishali Gupta, MS1, Amod Gupta, MS1, Ramandeep Singh, MS1, and Kusum Sharma, MD3
2
MD
,
1
2
Advanced Eye Center, Post Graduate Institute of Medical Education and Research, Chandigarh, India, Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India, and 3Department of Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
ABSTRACT Purpose: To compare the efficacy of devR and MPB64 PCR in the diagnosis of intraocular tuberculosis. Methods: Prospective, nonrandomized study. Seventy-five patients were enrolled in 3 groups. Group A had 25 patients with presumed intraocular tubercular uveitis, group B had 25 controls with specific uveitis other than tubercular uveitis, and group C included 25 non-uveitic negative controls. The undiluted vitreous/ aqueous samples were collected and subjected to PCR assay for devR and MPB64 gene sequence of Mycobacterium tuberculosis (MTB) to detect sensitivity and specificity. Results: devR PCR was positive in 16 (64%) out of 25 patients with presumed tubercular uveitis. MPB64 PCR was positive in 18 (72%) out of 25 patients with presumed tubercular uveitis. The sensitivity and specificity of devR were 64 and 100%, respectively. The sensitivity and specificity of MPB64 PCR were 72 and 100%, respectively. Conclusion: devR PCR is not a better tool than MPB64 PCR for diagnosing intraocular tuberculosis. Keywords: devR PCR, MPB64 PCR, polymerase chain reaction, retinal vasculitis, tubercular vasculitis
Diagnosis of intraocular tuberculosis (IOTB) in a tuberculosis (TB) endemic area poses a challenge due to diverse manifestations, its similarity to other uveitis entities, its paucibacillary nature, the difficulty in obtaining samples, lack of adequate samples, and the limitations of current diagnostic tests. Even though highly effective ATT treatment is available, this treatment has few adverse effects warranting some degree of certainty before starting it. It is difficult to establish the diagnosis of IOTB by histologic and microbiologic methods,1,2 and tuberculin skin sensitivity test (TST).3,4 Interferon-gamma release assays are still in primitive stage to diagnose IOTB.5 The polymerase chain reaction (PCR) technique has emerged as a powerful tool for rapid and accurate detection of the mycobacterial genome, with high specificity and a variable sensitivity.6–11
PCR is especially helpful for ocular fluids, as it can be performed with very small sample size. Most of the earlier studies have used IS6110 as a target specific for mycobacterium tuberculosis (MTb) for IOTB.9–15 However, absence or presence of very few copies of IS6110 has been reported in Indian population,12,13 which can result in missed cases suspected of TB. MPB64 gene-based PCR specific for MTB has been well evaluated for diagnosis of IOTB and has been found to be more sensitive than IS6110 PCR.14,15 The devR sequences are universally detected in MTB strains16,17,18 and it has been well evaluated in various extrapulmonary devR PCR has been shown to be highly sensitive and specific in the diagnosis of pulmonary and extra pulmonarytuberculosis.16–21 To the best of our knowledge, no study has earlier evaluated devR gene-based PCR for diagnosis of
Received 16 August 2014; revised 20 October 2014; accepted 22 October 2014; published online 26 November 2014 Correspondence: Dr. Ramandeep Singh, Associate Professor, Advanced Eye Center, Post Graduate Institute of Medical Education and Research (PGIMER), Sector 12, Chandigarh, India 160012. E-mail: [email protected]
47
48 P. Kataria et al. IOTB. Therefore, the present study was undertaken to evaluate the role of devR PCR in the diagnosis of IOTB and compared its efficacy with MPB64 PCR.
MATERIAL AND METHODS The study was conducted at our tertiary eye care center between July 2012 and November 2013. This study adhered to the guidelines in the Declaration of Helsinki. After institutional ethics committee approval and informed consent of the patient, we prospectively enrolled 75 patients into 3 groups in this study. Group A included 25 patients with presumed tubercular uveitis based on clinical signs and corroborative evidence.22 The diagnosis of TB was presumed when the patient had (a), signs of active uveitis such as cells/flare in the anterior chamber, with or without granulomatous keratic precipitates or iris nodules, iris granuloma, vitreous cells, snowballs, snow-banking, retinal vasculitis with/without perivascular choroiditis scars, with/without vitreous hemorrhage or tractional retinal detachment, serpiginous-like choroiditis, choroidal granuloma, or neuroretinitis; (b) corroborative evidence included positive tuberculin skin test (TST) according to the CDC recommendations (TST, 10-mm induration or more at 48–72 h) or positive Quantiferon-TB (QFT-TB) Gold test, or a positive chest x-ray; (c) all known causes of infectious uveitis or known noninfectious uveitis syndromes ruled out. Patients who were receiving anti-tubercular treatment (ATT) at the time of collection of vitreous fluid or received ATT in the past were excluded. Group B included 25 patients as positive controls, i.e. uveitis patients with other nontubercular etiologies, such as viral uveitis and endophthalmitis. Group C included 25 patients with noninflammatory ocular conditions, such as retinal detachment and macular hole, who were undergoing routing pars plana vitrectomy. No additional tests were done for this group of patients. All the enrolled patients underwent detailed ophthalmological examination, including best corrected visual acuity using the Snellen’s chart, intraocular pressure on Goldmann applanation tonometery, and slit-lamp biomicroscopy. Ancillary tests such as fundus photography, fundus fluorescein angiography, spectral domain optical coherence tomography, and ocular ultrasonography were performed as and when required. Group A patients underwent baseline uveitis investigations that included TST, chest x-ray, erythrocyte sedimentation rate, and Treponema palladium hemagglutination (TPHA) tests. In group B patients, investigations were ordered to diagnose the specific etiology, i.e. PCR for viral etiologies and smear and culture examination of the patients with endophthalmitis.
Sample Collection The aqueous humor was aseptically collected with a disposable 25- or 27-gauge needle on a tuberculin syringe under an operating microscope. The vitreous humor was aseptically collected at the beginning of 3-port pars plana vitrectomy and transferred to the laboratory in the cold chamber immediately. Our aim was to procure vitreous or aqueous cells in an ocular sample from the eye in cases other than vitreous hemorrhage.
Technique and Analysis of PCR The DNA was extracted from the fluid sample using a DNAse tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The extracted DNA specimens were stored at 20 C until used. The devR PCR was performed using the forward primer devR1 (50 -ATCTGTTGTCCCGCATGCC-30 ) and reverse primer devR2 (50 -GTCCAGCGCCCACATCT TT-30 ), which amplified a 162–base pair fragment of the devR single copy gene.21 The MPB64 PCR were performed using the forward primer MPB641 (50 -TCCGCTGCCAGTCGTCTTCC-30 ) and reverse primer MPB642 (50 - GTCCTCGCGAGTCTAGGCCA-30 ), which amplified a 240–base pair fragment of the MPB64 single copy gene.23 During DNA extraction and amplification, strict precautions were taken, including separate areas for DNA extraction, reagent preparation, amplification, and product detection, and regular meticulous cleaning of surfaces with 10% hypochlorite. In addition, all the reagents were aliquoted upon arrival in the laboratory. Positive and negative controls were included with each set of reactions. Positive control DNA extracted from H37RV was used and for negative control PCR grade water was used. Statistical Methods The sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the standard formula as follows (positive test: a = disease, b = no disease; negative test: c = disease, d = no disease): Sensitivity = a/(a + c) Specificity = d/(b + d) Positive predictive value = a/(a + b) Negative predictive value = d/(c + d)
RESULTS Group A comprised 25 patients with 20 males and 5 females. Group B had 15 males and 10 females, while group C had 25 negative controls with 18 males and 7 females. The mean age was 32.12 ± 10.27 Ocular Immunology & Inflammation
PCR for Intraocular Tuberculosis (range: 18–52) years in group A, 38.16 ± 19.64 (range: 8–70) years in group B, and 48.4 ± 16.81 (range: 5–68) years in group C. In group A, vitreous fluid samples were collected from 24 patients, i.e. vasculitis vitreous hemorrhage (16), choroiditis (7), intermediate uveitis (1), and panuveitis (1). Undiluted vitreous sample was taken at the start of the vitreous surgery as they all pars palna vitrectomy (PPV) for therapeutic or diagnositic vitrectomy. Therapeutic PPV was done in eyes with vitreoeus hemorrhage and diagnostic PPV was done in rest of the eyes. Aqueous fluid sample was collected from 1 patient with choroiditis. Vitreous fluid samples were collected from all the patients in groups B and C. In group A, all 25 patients had a positive tuberculin skin sensitivity test (induration of410 mm at 72 h) and 3 patients showed evidence of calcified lymph nodes on chest x-ray (Table 1). None of the patients had miliary TB. In group A, patients with presumed tubercular uveitis, devR PCR was positive in 16 patients and negative in 9 patients (Table 1, Figure 1). MPB64 PCR was positive in 18 patients and negative in 7 patients (Table 1, Figure 2). With devR PCR, we found sensitivity and specificity of 64 and 100%, respectively. With MPB64 PCR, we found sensitivity and specificity of 72 and 100%, respectively. Both PCRs were simultaneously positive in 14 cases out of 25. In 6 of the 25 patients, both PCR results were opposite. Five cases were negative for both devR and MPB64
49
PCR (Tables 2, 3). Combined (at least one positive) sensitivity was 80% and specificity was 100%. In control groups B and C, ocular fluid samples were negative in all patients for both devR PCR and MPB64 PCR (Table 3). There were no false-positive cases. Statistically significant difference was found in terms of difference in PCR results in cases and control groups (p5.001). In subgroup analysis of group A, vasculitis with vitreous hemorrhage subgroup, of the 16 patients, 12 were positive for MPB64 PCR and 9 were positive
FIGURE 1. DevR PCR Gel picture: L1- MM, L2- Positive control with bending pattern on gel, l3-Negative control: L4-L6 Positive clinical samples, L7-8; negative samples.
TABLE 1. devR PCR and MPB64 PCR results in group A. S no
Mantoux test
Chest x-ray
devR PCR
MPB64 PCR
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
Normal Abnormal Abnormal Abnormal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Negative Negative Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Negative Positive Positive Positive Negative Positive Positive Negative Positive
Negative Negative Positive Positive Negative Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive
!
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Diagnosis Vasculitis with vitreous hemorrhage Panuveitis Choroiditis Choroiditis Intermediate uveitis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Choroiditis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage Choroidal granuloma with choroiditis Choroiditis Vasculitis with vitreous hemorrhage Vasculitis with vitreous hemorrhage
50 P. Kataria et al.
FIGURE 2. MPB64 PCR Gel Picture: L1-MM, L2-Positive control, L3-L4, L6: Positive patient DNA, L5-Negative control, L7, 8-Negative patient DNA.
TABLE 2. Detailed results of positive PCR in group A patients. Specific genome results (Group A = 25 samples) devR positive alone MPB64 positive alone Both devR and MPB 64 positive Either devR or MPB64 positive
Number of samples (%) 16 18 14 20
(64%) (72%) (56%) (80%)
for devR PCR. MPB64 PCR was more sensitive than devR PCR (75 vs. 56.25%, respectively) in this subgroup. In the choroiditis subgroup, of 7 the patients with active choroiditis, all were positive for devR PCR (sensitivity 100%) and 6 were positive for MPB64 PCR (sensitivity 85.7%). One patient of choroiditis had co-existent choroidal granuloma that was both devR and MPB64 PCR positive (Table 4).
DISCUSSION Nucleic acid amplification techniques (NAAT), such as PCR, are a robust method for diagnosis of paucibacilliary extrapulmonary tuberculosis. devR PCR has been used to diagnose extraocular tuberculosis, with sensitivity ranging from 46.7 to 98%16–21 and specificity ranging from 66.7 to 100%.16–21 There is no available study in literature regarding the use of devR PCR for diagnosis of IOTB. Halder et al.20 demonstrated high sensitivity (87.6%) and specificity (92%) of devR PCR in the diagnosis of tubercular meningitis (TBM). Chakravorty et al.,16 in their study with devR PCR, demonstrated that sensitivity ranged between 46.7 and 75.5% and the specificity ranged between 66.7 and 100% in three groups of specimens (pleural fluid, pleural tissue, and lymph node). Hallur et al.19 compared IS6110 with devR PCR in cases of
abdominal tuberculosis and showed a higher sensitivity of 80% with devR PCR as compared to 77% with IS6110 PCR. In the present study, devR PCR was found to be highly specific in the diagnosis of IOTB, corroborating the results of previous studies on extraocular tuberculosis, but it lacked sensitivity in detecting intraocular tuberculosis, similar to previously reported studies from other extrapulmonary sites.16–20 In the present study, MPB64 PCR (72%) was found to be more sensitive than devR PCR (64%) in diagnosing IOTB. However, when any of the two gene targets (MPB64, devR) were used, sensitivity for diagnosis of IOTB increased to 80%. Earlier studies using MPB 64 gene target for diagnosis of IOTB have shown sensitivity ranging from 47.8 to 66.66%.14,15,24 A majority of the PCR assays evaluated for diagnosis of TB have targeted the IS6110 gene, as there are multiple copies of IS6110 in the MTb genome (1–25 copies/genome), resulting in higher sensitivity. However, it has been shown that some Indian strains of MTb do not contain IS6110 or have low copy numbers of this gene, thus missing these cases.13 So, there is need to evaluate other targets for rapid diagnosis of ocular tuberculosis as recently published by Balne et al.25 and Sharma et al.15 IS6110 has been used for the diagnosis of IOTB extensively. When compared to previous studies,10,11,23 sensitivity of devR PCR (64%) was greater in our study. The sensitivity of IS6110 PCR is reported between 37 and 58.82%.10,11,23 MPB64 has been reported as 10,000 times more sensitive than IS6110 for the diagnosis of tubercualr uveitis.14 Our study supports this finding. Although clinically strong suspicious cases of tubercular uveitis were enrolled in group A, some turned out to be negative with PCR. So testing for one particular gene may give false-negative PCR result. To overcome this, a novel multitargeted PCR technique18 has been introduced. Sharma et al.18 reported high sensitivity (77.77%) with multitargeted PCR (IS6110, MPB64, protein b) as compared to individual PCR. This approach would be helpful in the diagnosis of the presumed tubercular uveitis with increased sensitivity. In the present study, a combination of two PCR revealed sensitivity of 80%, when either of the two PCR (devR and MPB64) was positive. Hence, these two have the potential to be used for multitargeted PCR. In subgroup analysis, in patients with the vitreous hemorrhage subgroup, we found the sensitivity of MPB64 (75%) to be greater than that of devR PCR (56.25%). For the diagnosis of Eales’ disease using various PCR primers, previous studies had reported sensitivity of 41.6% (Biswas et al.,11 IS6110), 47.8% (Madhvan et al.,14 MPB64), and 57% (Singh et al.,22 MPB64). In choroiditis patients, sensitivity of devR PCR was 100%, while it was 85.7% for MPB 64. Ocular Immunology & Inflammation
PCR for Intraocular Tuberculosis
51
TABLE 3. Sensitivity and specificity of devR and MPB64 PCR in group A patients. Test devR MPB64 Combined (at least one positive)
Test results
Group A (n = 25)
Groups B and C (n = 50)
Positive Negative Positive Negative Positive Negative
14 11 16 9 20 5
– 50 – 50 – 50
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
64
100
100
84.75
72
100
100
87.71
80
100
100
90.90
PPV, positive predictive value; NPV, negative predictive value.
TABLE 4. Subgroup analysis of group-A patients. Etiologies (n = no. of patients)
devR positive (%)
MPB64 positive (%)
9 (56.25) 7 (100) – –
12 (75) 7 (85.71) – –
Vitreous hemorrhage (16) Choroiditis (7) Intermediate uveitis Panuveitis
Gupta et al.23 reported IS6110 PCR positivity in 4 of the 5 eyes of choroidal tuberculosis simulating serpiginous choroiditis. The study was marred by the small number of patients; larger studies could further show the promising role of devR PCR in eyes with choroiditis. Our study has a few limitations, the major one being the small number of patients. In groups B and C, patients were presumed to be nontubercular in origin and investigations related to uveitis workup were not considered necessary in them. The follow-up of patients and assessment of clinical response to treatment was not carried out in this study. There are some known inherent limitations of PCR, i.e. PCR negativity in a few clinically suspected intraocular TB cases, due to the presence of the low number of bacteria or poor lysis of bacteria in the ocular fluids. Sometimes the tough cell wall of M. tuberculosis makes the isolation of target DNA difficult or it could be affected by the presence of inhibitors in the sample. TST and QFT-TB were not carried out in group B and C patients. This could be one of the limitations and future studies can be planned taking these factors in consideration. Use of clinical diagnosis of presumed intraocular TB as the gold standard has its own issues. Culture/microscopy was not used due to inherent problems of low sensitivity and prolonged turn around time. The alternative of favorable response to anti-tubercular treatment in presumed tubercular uveitis was not used in this study. Despite the above limitations, our results clearly show that MPB64 PCR is is amore sensitive diagnostic test for IOTB in comparison to devR PCR. Further, this study for the first time shows the role of devR PCR in the diagnosis of IOTB. The role of devR needs to be !
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evaluated in a large sample size, especially in IOTB patients with ocular finding of choroiditis.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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